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Interaction between the complement degradation product C3bi and leukocyte integrin alphaMbeta2, which are critical for host defense against foreign pathogens and in tumor cell surveillance is analyzed by a detailed mapping of the C3bi binding site. The results support a mosaic model, in which different ligands engage different amino acids to bind alphaMI-domain, accounting for the broad recognition capacity of integrin alphaMbeta2.

The interaction of opsonized particles with neutrophils and monocytes is influenced by the level of reaction that takes place between the receptor and the major integrins of neutrophils. A binding assay performed on 293 human kidney cells proved that the C3bi binding pocket of amino acids are composed of the P147-R152, P201-K217 and K245-R261 regions, which are all instrumental in facilitating the alpha (sub m)beta (sub 2) binding. It was also shown the formation of this bond may be hindered by induction of conformational changes or direct competition for the ligand binding site.

The availability of the amino acid sequence, CS-1, in plasma fibronectin (Fn), was tested by raising an antibody to the synthetic peptide CS-1. The CS-1 sequence was discovered to be minimally exposed in plasma Fn and immobilization of Fn, a model matrix deposition, resulted only in a small increase in its exposure. However, digestion of Fn with selected proteases caused substantial expression of the CS-1 sequence. Findings indicated that exposure of the CS-1 sequence in plasma Fn by proteolysis with cathepsins D and B may have a regulatory role in cell adhesion.

The main binding site of human coagulation factor IX at the bovine aortic endothelial cell receptor is situated among the amino-terminal 14 amino acids of its light chain. This binding quality can be completely transferred to protein C. The study confirms the technique of using strategically generated chimeric recombinant molecules to indicate the areas of proteins' functional properties. It also permits new coagulation proteins to be constructed that have others' properties.

The cell-binding domains of fibronectin vary depending on the conformation of the protein. Experiments with monoclonal antibodies to two cell-binding domains show that the antibodies impaired platelet adhesion to fibronectin. In solution, two ninth type III repeats and one tenth type III repeat were expressed. However, when fibronectin was adsorbed onto polystyrene, a second tenth type III repeat was expressed.

Tb3+ luminescence spectroscopy of platelet integrin GPIIb-IIIa (alpha(IIb)beta3) to identify its cation-binding site reveals that five cation binding sites are present on the intact GPIIb-IIIa receptor and they exhibit unequal binding affinity to Tb3+. Tb3+ also binds the divalent Mn2+ and Ca2+ cations that participate in the ligand binding activity of the GPIIb-IIIa receptor, while the peptides related to the GPIIb Ca2+-binding sequence mediate the cation-Tb3+ binding. The 118-131 GPIIIa sequence is more potent in binding divalent cations to Tb3+ than the 208-221 sequence. The first coordination position in the amino acid terminal is essential for effective cation-synthetic peptide binding.

A comparison of the binding properties of kringles of plasminogen and apolipoprotein of lipoprotein (Lp(a)) reveals their similarities in binding lysine and lysine analogs. Lp(a) contains lysine binding sites involved in binding with fibrin and cells. The binding properties of Lp(a) and kringles are similar but not identical, and the binding sites of the two are distinct.

The changes in plasminogen binding proteins on surfaces of leukocytes are studied using phorbol myristate acetate simulation of U937 monocytoid cells and culture of human peripheral blood neutrophils as model conditions for modulation of plasminogen binding. The outcome leads to identification of a new regulated plasminogen receptor on these cell types, which play a vital role in the changes in plasminogen binding capacity of the cells.

Integrin [alpha.sub.IIb][beta.sub.3] is used as a model to show that a previously reported activating mutation [alpha.sub.IIb]{[R.sup.995]D} that perturbs the electrostatic interface in the clasp only partially activates [alpha.sub.IIb][beta.sub.3] and that extensive activation of the receptor is achieved by complete deletion of [alpha.sub.IIb] cytoplasmic tail (CT). The results have provided the molecular insights into how distinct regions of the cytoplasmic tails differentially and cooperatively regulate integrin activation.

Interactions between the complement degradation product C3bi and leukocyte integrin αMβ2 are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the αMI-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the αMI-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding αLl-domain residues, Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213,I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-1215, in C3bi recognition. Furthermore, the αDI-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the αDI- domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the αMI-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to αMI-domain, accounting for the broad recognition capacity of integrin αMβ2. [ABSTRACT FROM AUTHOR]

Examines the molecular basis of C3bi recognition by alphaMbeta2 receptor, the major integrin on neutrophils. Utilization of a series of mutant receptors; Structural similarities and functional limitations of mutant receptors; Composition of C3bi binding pockets; Role of K245 region in mediating C3bi binding; Mechanisms of monoclonal antibody blocking.

The short cytoplasmic tails regulate activation of integrin adhesion receptors via clasping/unclasping of their membrane-proximal helices. Using integrin alpha(IIb)beta(3) as a model, we show that a previously reported activating mutation alpha(IIb)(R(995)D) that perturbs the electrostatic interface in the clasp only partially activates alpha(IIb)beta(3) and that extensive activation of the receptor is achieved by complete deletion of alpha(IIb) CT or triple mutations in alpha(IIb)(V(990)A/F(992)A/R(995)D) that disrupt both electrostatic and hydrophobic interfaces in the clasp. The results provide quantitative evidence for an equilibrium-based integrin activation process where shifting the equilibrium to the fully activated state requires total unclasping of the cytoplasmic tails. We further demonstrate that while the C-terminal region of the alpha(IIb) tail minimally influences alpha(IIb)beta(3) activation, the C-terminal region of the beta(3) tail is critically involved. A disease-causing mutation of S(752)P in this region, but not S(752)A, suppressed partial activation induced by R(995)D or the talin head domain but did not affect activation induced by alpha(IIb) truncation. NMR spectroscopy revealed that S(752)P but not the S(752)A mutation disrupted a C-terminal helix within the beta(3) tail, suggesting that the C-terminal helix may regulate the equilibrium-based clasping/unclasping process. Together, these data provide molecular insights into how distinct regions of the cytoplasmic tails differentially and cooperatively regulate integrin activation.